ABCA4 is an ABC transporter found in retinal photoreceptor cells. Mutations in ABCA4 have been implicated in retinopathies such as autosomal recessive Stargardt macular degeneration, cone-rod dystrophy, retinitis pigmentosa and age-related macular degeneration. ABCA4 functions as an importer, and ?flippase?, moving N-retinylidene-phosphatidylethanolamine (N-retinylidene-PE) from the lumen to the cytoplasmic leaflet of disc membranes. Previous studies indicate that mutations in ABCA4 profoundly influence this flippase activity, resulting in a buildup of toxic retinoid substrates in the lumen. A potential approach for treatment of Stargardt Disease is to correct these functional defects with small molecule transport modulators. This approach is analogous to the use of small molecule ?potentiators? such as Kalydeco (Vertex Pharmaceuticals) to successfully correct channel gating defects in the G551D CFTR mutant in Cystic Fibrosis. The discovery of ABCA4-directed therapeutics to treat retinal disease requires development of novel assays to measure restoration of transporter function for ABCA4 disease mutants. Moreover, these assays must be scalable to screen large (e.g..50K to > 1M) small molecule compound libraries. Our specific aims are toestablish proof-of- concept for a robust high-throughput screening (HTS) platform for ABCA4-directed drug discovery. First, a biochemical platform must be developed for assessment of the functional and structural consequences of common disease variants of ABCA4. Mammalian cell expression protocols will be established for wild-type ABCA4 and a panel of ABCA4 disease mutants. Purification and reconstitution of ABCA4 into liposomes will then be optimized for functional assay development. Existing literature lipid transport assays measure active transport of ABCA4 substrates, such as radiolabeled N- retinylidene-PE, or fluorescent lipids, from the membrane inner leaflet to the outer leaflet. These assay protocols are unsuitable for high-throughput screening (HTS) of small molecule libraries, as they rely either on a cumbersome centrifugation step to separate donor proteoliposomes from acceptor liposomes, or multiple quenching steps and detergent addition, which would introduce significant error and impose technical constraints in a high-throughput setting. To address these limitations, we will use a simple phospholipid tagging strategy to allow separation of donor proteoliposomes from acceptor liposomes in a high-throughput format. We will establish protocols in which biotinylated lipids are incorporated into acceptor liposomes, allowing these liposomes to be tethered to streptavidin coated substrates on multi-well filter plates commonly used in HTS. After a wash step to remove the proteoliposomes, the acceptor can then be measured by either scintillation proximity methods with a radiolabeled substrate, or total fluorescence for a fluorescent phospholipid substrate.